PROJECT SUMMARY The central goal of our proposed research is to understand the functional consequence of polycyclic aromatic hydrocarbon (PAH) exposure, specifically benzo[a]pyrene (B[a]P), during development on the gut microbiome and liver and intestinal tissues, and how these functional alterations may impact the susceptibility for disease onset. We bring a function-based approach that is irrespective of microbiome composition, and targets microbiota and host cell and enzyme activities directly. Thereby, we directly measure functional capacity, not infer it from other measurements. We will demonstrate how cell and enzyme level functional capacities can be used as markers for disease susceptibility due to PAH exposure. PAHs represent a potential carbon and energy source for gut microbes, but it is unclear how the microbiota interact with PAHs, and how microbe-mediated PAH metabolism may alter bacterial functions or composition, and/or contribute to the negative consequences of PAH exposure. Additionally, it is unclear how metabolic activities at the host-gut microbiome interface respond to PAH exposure, and how the interaction of enzymatic activities may contribute to disease outcome following developmental PAH exposure. We will make a significant functional advance in the understanding of microbial metabolic functions and host-gut microbiome enzymatic activities, and how they influence disease onset due to PAH exposure in development, by applying an activity-based protein profiling (ABPP) platform for quantification and characterization of taxa and enzyme functional activities. By being able to identify the functionally active cells and enzymes within the microbiome, meaningful conclusions can be drawn about the connection between the gut microbiome and metabolic activities, and how they are perturbed by PAH exposure. Our ABPP platform will deploy chemical probes that target only catalytically active enzymes. The probes target the functional enzymes, but will enable isolation, characterization, and quantification of both the cells expressing those functions, and the specific enzymes catalyzing metabolism in the gut, and quantify enzyme activities within host tissues. With our ability to make measurements of functional capacity, we will address the central hypothesis that developmental exposure to the potent PAH, B[a]P, profoundly impacts the functional capacity of metabolism in host liver, intestine, and the gut microbiome resulting in significant inflammation and an environment with heightened susceptibility to PAH- mediated toxicity and tumorigenesis. We will initially develop new activity-based probes to add to our existing suite for targeting gut microbe and host tissue enzyme activities. We will then determine the microbiome's contribution to the host metabolic response to B[a]P exposure and define host endpoints of exposure toxicity. Conventional and germ-free mice model studies will be employed to parse out microbiome-induced host responses from host-only responses. After determining how the gut microbes and host tissues respond to exposure, and the functional role of the microbiome, we will evaluate direct oral exposure to B[a]P versus maternal-to-child transfer via lactation. Finally, we will look at the long-term impacts of B[a]P exposure and show how early life exposure leads to enhanced susceptibilities for PAH-induced diseases, including tumor formation and intestinal and liver inflammation. Completion of this project will provide an unparalleled view of functional capacity in the gut microbiome, how it changes as a result of PAH exposure, and how this leads to heightened susceptibilities. We anticipate that the known toxicity and carcinogenicity caused by PAHs involves regulation by the gut microbiome that heightens susceptibility if exposures occur during host and microbiome development. Finally, we will demonstrate that microbiome composition is not an adequate predictor of PAH-induced disease susceptibility, but functional capacities at the cell and enzyme level can be viable markers.